PROJECT SUMMARY/ABSTRACT Metallo-b-lactamases (MBLs) are bacterial enzymes that render pathogenic bacteria resistant to the largest class of antibiotics. Although a number of MBL inhibitors have been published, clear structure-activity relationships (SARs) are often difficult to discern, and none of these inhibitors has advanced to clinical use. We propose that the disconnect between the discovery of inhibitors and the development of clinically-useful compounds is due, in part, to a lack of understanding the mechanisms whereby these compounds work, and by the predominance of metal-stripping mechanisms, which are inherently non-specific and have associated toxicity issues. To directly address these issues, we propose to discern the mechanism of inhibition of several under-defined MBL inhibitors. Specific aim #1 will characterize inhibition mechanisms of several MBL inhibitor classes. Many reported MBL inhibitors contain functional groups with potential to bind metal ions, but their mechanisms remain undefined. Here, we use a multidisciplinary approach to determine if selected classes of MBL inhibitors work by metal- stripping, by ternary complex formation (inhibitor:metal ion:protein) or by other mechanisms. This information will enable more effective SAR studies and allow better selection of candidates more suitable for clinical applications. Specific aim #2 will develop two novel techniques to probe inhibitor binding to MBLs. One technique is EPR- based and is predicted to yield information about an invariant b-hairpin loop that resides over the MBL active sites. The second technique is native MS-based and is predicted to yield direct information about the mechanism of inhibition using lower concentrations of enzyme/inhibitor. The long-term goal of this research program is to identify the most promising scaffolds for MBL inhibitors, which can be given in combination with existing b-lactam containing antibiotics to treat antibiotic resistant bacterial infections. Through clearly determining the mechanism of action of promising MBL inhibitor scaffolds, we will provide a clearer pathway for these inhibitors to proceed to clinical trials.